Preparation of thio-alkyl pantothenamides



Patented Jan. 13, 1953 UNITED STATES PATENT OFFICE PREPARATION OFTHIO-ALKYL PANTOTHENAMIDES Esmond E. Snell, Madison, Wis., and Eugene L.

Wittle and James A. Moore, Detroit, Mich., assignors to Parke, Davis &Company, Detroit, Mich., a corporation of Michigan No Drawing.

Application March 28, 1951, I

Serial No. 218,096 7 Claims. (01. 260561) This invention relates tomethods for obtaining organic sulfur compounds. More particularly, theinvention relates to methods for obtaining organic sulfides having theformulas,

In accordance with the present invention;'the

organic sulfides can be prepared by reaction of an ester of pantothenicacid with hydrazine forming pantothenyl hydrazide. The latter is thenallowed to react with nitrous acid to form the corresponding pantothenylazide. The azide is treated with fl-mercaptoethylamine or withbis(;3-aminoethyl)disulfide to obtain the sulfides I and II,respectively.

The above reactions are illustrated by the following:

' 2 where R is an alkyl, a lower phenalkyl or phenyl radical.

In carrying out the preparation of the hydrazide, hydrazine, which canbe furnished to the reaction mixture in the form of hydrazine hydrate,hydrazine sulfate, or anhydrous hydrazine, is allowed to react with anester of pantothenic acid in the absence of or the presence of a.solvent such as water, a lower aliphatic alcohol, or

amixture of same, or an excess of hydrazine; itself. Thereactiontemperature' is maintained at about 0 C. to 100 C.

In the formation of the azide of pantothenic acid, the pantoth'enic'acidhydrazide is allowedto react with nitrous acid in the presenceof'w'ater, lower aliphatic alcohols, mixtures of water andwater-immiscible lower aliphatic alcohols, such as butanol, and mixturesof water and lower molecular weight fatty acid esters such as ethylacetate and the like. In the case of two-phase systems such as thelatter, the azide product occurs in the organic solvent phase for themost part. The nitrous acid may be generated from an inorganic nitriteand a strong acid or it can be generated from the reactionof an organicI CH20H-C-CH OH-C ONH+CH:CHa-CONHNH: ROH

CHa CHzOH-JJ-CHOH-CONBF-CHr-CHa-CON: 21110CHzOH-(J-CHOH-CONH-CHzCHz-CONH-CHa-CIIa-SH 4- EN! nitrite such as thealkyl nitrites, e. g. butyl nitrite, and an inorganic or organic acid.During the reaction of the hydrazide with the nitrous acid, thetemperature is maintainedbelowabout 15 C. and an excess of dilute orconcentrated acid 5 is desirable.

The pantothenic acid azide, when formed in 1 the aqueous or aqueous andwateremiscibleore. ganic solvent mixture, is preferablyused directly, inthe solution for the next step. When the twophase system is employed, itispreferabletonse the organic layer containing the azide in the nextstep.

The reaction of the pantothenic acid azide and B-mercaptoethylamine orbistB-aminoethyl) -15.

disulfide can be carried out under a varietygoi-r conditions. When theazide is formed in the single phase such as the water orwater-alcohol-solution, the reaction is carried out by adding theappropriate sulfur compound and then adjusting the pH of the solution tobetween about 8 to :13 byaddition'of a basic solution such as an"aqueous alkali metal hydroxideor carbonate or,. alter-:- nativelyg-anexcess of the amine reactant. When: theazide is collected in the-organicphasezof-the g5 two-phase system described 'above,the.reaction with theappropriate "sulfur. :compoundis carried-v out bymerely addinganzexcessof the latterz" The: reaction temperatures .in eithertcaseimaytranger: from 0"-to- 60. CJ,".butipreferahlyzfrom about 20 to C.

Whilenormention was :made of -the--stereoisomeric form of thepantothenic-acid esters usedin thezabove reactions; it is to-beunderstood thatthe rscope'pof the present invention: clearly ini 5cludes boththe esters of the dextrorotatory-biologically activend-pantothenic acid, as .well :as those-of the opticallyinactive-dl-pantothenic acid in;which-1latter1case the opticallyinactive sulfide, compounds-are obtained as;-products. 49

The :inventionzis; illustrated bythe. following examplcsn.

Example -1 To 11 g. of hydrazine hydrate (85%) cooled in an ice bath isadded 11.5 g. of methyl d-panto- 5 thenate and the cold mixture isstirred vigorously After the reaction takes place and the mixture iswarmed to 30 0., it is allowed to stand at room temperature-for=two-,days and .then. .evan-r orated to dryness in vacuo at 50 C. Theresidue (14.7 g.) of pantothenyl hydrazide is a clear glassy oil.

To 7.3 g. of this crude hydrazide dissolvedcin... 21 cc. of warm wateris added 4 cc. of glacial acetic acid. The solution is placed in abeaker and cooled in an ice bath and, .withstirringa solu-. tion of 1.7g. of sodium nitrite in 5 'cc. of water is added dropwise. One cc. moreof acetic acid is added to maintain the pH at 5 and the solution isallowed to stir in the cold for one hour... .To-the. solution is addedsufficient strong sodium hydroxide solution to adjust the pH to 8 and 2g. of 8- mercaptoethylamine is added. The pH isadjuste ed to 9 withadditional sodium hydroxide and the solution is stirred in the cold forone hour and then at room temperature for one hour. The pH of thesolution is then adjusted to '7 with dilute acid and the resultingneutral solution is... concentrated in vacuo to a clear, colorlesviscous oil containing N- E -pantothenyll -;3-aminoethanethiol in acrude state.

The crude N-[(+) -"-pantothenyl]-B-amino-' ethanethiol so obtained ispurified by dissolving the crude reaction product in 45 cc. of anhydrousn-butanol and pouring the resulting solution 4 through a chromatographcolumn containing 272 g. of activated carbon. The column is washed withn-butanol and fractions are collected from time. toptime and thefractions containing solids assaying about 25. :to 40% pureN-[(+)-pantothenyl]-,8-aminoethanethiol against Lactobacil- Zus:helveticus 80 poured onto a chromatograph column containing 136 g. of analkaline earth aluminunr'silicate known commercially as Superfiltrol.The column is washed thoroughly with anhydrous n-i-butanol and thewashings and main solution discarded. n-Butanol saturated with water ispoured through the column to elute the N-[-(+) -pantothenyll-;8aminoethanethiol and .the..resulting..,solution evaporated to dryness inva'cuo.- at low temperature to obtain the desired product in pure form.Its formula is,

Instead of pouring the anhydrous n-butanol solution onto the. alkalineearth aluminum silicate chromatograph column, one can simply repeatthetreatment with a carbonchromatograph column to obtain the pure product.In some instances, the first carbon treatment produces fractionscontaining pure N-[ -pantothenyll-' fi-Laminoethanethiol-and in thosecases it is,. of course, not necessary to treat the fraction withalkaline earth aluminum .silicate nor -again with activated carbon.

While the thiol compound "described" above is the chief product formedin the above reaction and purification; there may be a certain'amount ofthe corresponding disulfide occurring in the final product due tooxidation of .the thiol taking place-during the .purificatiomsteps;

lt'scamplev 2 To a solution of 4.8 g. of methyl d-pantothenatei in 10ccrof'methanol-is added 2.3 cc. of hydrazine hydrate The solution isallowed toistand at 25 C. for twenty hours and then evaporated todryness. inyacuo. at..50 to. leave a clear glassy oil. This is dissolvedin methanol and re-evap oratedseveral times to remove excess hydrazine.The d-Tpantothenyl hydrazide is dissolved in 25 cc; ofwatenin a beakerand cooled in an ice bath and with stirring is acidified to pH 3.5 with6 N hydrochloric acid. A solution of 1.4 g. of sodium nitrite. in 7 .5.cc. of Water is added slowly, keeping the pl-1 at 3.5 with more acid."The solution is ..allowed to stir in the cold for one hour and then'made basic to pH 8 with 2 N sodium hydroxide solution A solution of 1.6g. of fi-mercaptoethylamine in 10 cc. of water is added and afterstirring in the cold for twenty minutes the solution is allowed to standat 25? C. overnight. The solutionwas acidified from"pH'8.5 to 7.3 andconcentrated in vacuo to a clear colorless viscous oil. The pure productcan be isolated from this oil by the method described in Example 1.

Example 3 To 7.3 g. of crude d-pantothenyl hydrazide dissolved in 21 cc.of water and stirred in a beaker cooled onan ice bath 'isadded'sufii'cientfi Nhy-' drochloric acid to shift the pH to 4. Then asolution of 1.7 g. of sodium nitrite in 5 cc. of water isadded-dropwise-over aperiod of one hour, keeping the pH at 4 by.additions of 6 N hydrochloric acid. After stirring for one-half hour,2.8 g. of bisQS-aminoethyDdisulfide dihydrochloride is added. The pH isthen adjusted to 8.5 with 50% aqueous sodium hydroxide solution and thesolution allowed to stir for one and one-half hours. It is thenacidified to pH 7.5 and concentrated in vacuo to a clear colorlessviscous oil. The pure product can be isolated from this oil by themethod described in Example 1. The formula of this product is,

[cmon-o-H0n-o-NH-omomd-Nmomonzs] CH3 2 Example 4 To a solution of 10 g.of methyl-d-pantothenate in 10 cc. of methanol is added a solution of 11g.., of 85% hydrazine hydrate in 10 cc. of methanol.* The solution isallowed to stand at 25 C. for sixteen hours and then gently refluxed forone hour. It is evaporated to dryness in vacuo at 50? C., redissolved inmethanol and evaporated several times to yield 13.4 g. of d-pantothenylhydrazide as a clear, glassy oil.

3 g. of this crude hydrazide is dissolved in 25 cc. of absolute ethanoland 15 cc. of 1.5 N hydrochloric acid in ethanol is added. Some white,solid hydrazine monohydrochloride precipitates and is filtered off anddiscarded. The clear filtrate is acidified with 5 cc. more 1.5 Nhydrochloric acid and then cooled in an ice bath. To the cold solutionis added slowly and with stirring 1.5 cc. of n-butyl nitrite. Thesolution is kept cold for forty-dive minutes and then 1.5 g. ofB-mercaptoethylamine is added and the solution made alkaline with 2 Nsodium hydroxide solution to pH 10. The solution is allowed to stand at25 C. forsixteen hours and then 15 cc. of water is added to dissolve allinsoluble solids and give a clear solution having a pH of 9.5. After onehour 6 N hydrochloric acid is added to pH 7 and concentrated in vacuo toa clear colorless viscous oil containing 36,210,000 LBF unitscorresponding to a yield of 75% of theory of the desired product. Thepure product can be isolated from this oil by the method described inExample 1.

Example 5 To 2.5 g. of crude d-pantothenyl hydrazide in 11 c. ofabsolute ethanol is added 15 cc. of 1.5 N hydrochloric acid in absoluteethanol. The White insoluble precipitate is filtered off, the clearfiltrate cooled in an ice bath, and 1 cc. of n-butyl nitrite added withstirring. After stirring for two hours in the ice bath, the solution of1 g. of fi-mercaptoethylamine in 5 cc. of water is added and then 5 cc.more water added to form a clear solution. The pH of the solution isadjusted to 9 with dilute sodium hydroxide and allowed to stand sixteenhours at 25 C. Dilute acid is added to pH 7 and the solutionconcentrated in vacuo to a clear colorless viscous oil containing14,400,000 LBF units corresponding to a yield of 36% of theory of thedesired product. The pure product can be isolated from this oil by themethod described in Example 1.

Example 6 To a solution of 2.5 g. of crude d-pantothenyl hydrazide in 11cc. of ethanol is added 30 cc. of water and 40 cc. of n-butyl alcoholand the mixture is placed in a beaker and cooled in an ice bath. Withstirring the solution is acidified with 6 N hydrochloric acid to pH 4and then a solution of 700 mg. of sodium nitrite in 5 cc. of water isadded. The pH is kept at 3.8-4 with acid and the solution stirred in thecold for forty-five minutes. The cold butanol layer is then separatedand to it is added 1 g. of fi-mercaptoethylamine. The pink color whichforms soon fades and the colorless solution is allowed to stand at 25 C.for sixteen hours. Microbiological assay showed 17,000,000 units of LBFactivity, or a yield of 41% of theory of the desired product. The pureproduct may be isolated from this solution as in the previous examples.

Example 7 To a solution of 2.5 g. of crude d-pantothenyl hydrazide in 11cc. of ethanol is added 6 cc. of 3 N I alcoholic hydrochloric acid andthe solution filtered from the insoluble hydrazine mono-hydro chloridewhich forms. The clear filtrate is mixed with 30 cc. of water and 40 cc.of butanol and cooled in an ice bath. The pH of the solution is I 2.8.With stirring a solution of 700 mg. of sodium nitrite in 10 cc. of wateris added slowly and the pH kept at 3 by the addition of dilute acid. Thesolution is stirred for one hour in the ice bath and the butanol layerseparated. .A second solution is prepared as follows: 230 mg. ofmetallic sodium is dissolved in 10 cc. of methanol and to it is added asolution of 1.12 g. of bis(;8-aminoethyl) disulfide dihydrochloride in10 cc. of methanol. The butanol layer above is washed once with 20 cc.of cold water and then added to the bis(e-aminoethyl) disulfidesolution. After standing for sixteen hours at 25 0., the solution isconcentrated in vacuo to a clear colorless viscous oil containing12,500,000 LBF units of activity which corresponds to 30% of thetheoretical yield of the desired product. The pure product can beisolated from this oil by the method described in Example 1.

Example 8 To a solution of 78.5 g. of ethyl-d-pantothenate in 100 cc. ofmethanol is added a solution of 78.5 g. of hydrazine hydrate in 50 cc.of methanol. The solution becomes warm due to reaction and is thenallowed to stand at 25 C, for sixteen hours. The solution is evaporatedin vacuo at 0.5 mm. pressure and 50 C. to leave 108.5 g. of clear,colorless oil, the hydrazide of d-pantothenic acid still containing somehydrazine.

The hydrazide is dissolved in 200 cc. of absolute ethanol, cooled in anice bath and to it is added 250 cc. of 3.3 N ethanolic hydrochloricacid, suflicient to make the solution test acidic. The white solidhydrazine monohydrochloride is filtered oflz and washed with a littleethanol. The clear filtrate is added to a mixture of 1200 cc. of waterand 1600 cc. of n-butanol and the whole mixture stirred and cooled in anice bath to 5-10 C. A solution of 22 g. of sodium nitrite in 50 cc. ofwater is added dropwise over a period of forty-five minutes at 8 C. 30cc, of 6N hydrochloric acid isalso added slowly to maintain the pH ofthe solution at 3.5-4. The solution is allowed to stir at 5 C. for twohours. The butanol layer is then separated from the water layer andWashed once with 200 cc. of ice water.

To it is then added a solution of 40 g. of B-mercaptoethylamine in cc.of methanol. The light red color which forms fades after one hour. Theaqueous layer above is extracted once with 500 cc. of butanol and thebutanol washed with 100 cc. of ice water and added to the main butanolreaction mixture. This solution is allowed to stand at 25 C. for sixteenhours and then concentrated in vacuo at 40 C. to 1180 cc. of butanolsolution. Microbiologicalassay shows 624,574,000

accepts 2 units of LBF activity which is 39% ofthetheoretical yield ofthe desired product.

The product in the butanol solution may be purified by passing it overan activated charcoal column containing 40 g. of activated charcoal foreach gram of crude product (obtained by drying and weighing a portion ofthebutanol solution) or by passing it over a column containing 50 g. ofalkaline earth aluminum silicate per gram of crude product, as inExample 1.

What we claim is:

1. Process for obtaining a member of the class consistin of a compoundof the formula,

which comprises reacting an ester of pantothenic acid with hydrazine toobtain the hydrazide of pantothenic acid, reacting said hydrazide withnitrous acid to obtain the azide of pantothenic acid, and thereafterreacting said azide with a member of the class consisting ofS-mercaptoethylamine and bis(c-aminoethy1) disulfide.

2. Process for obtaining a member of the class consisting of a compoundof the formula,

which comprises reacting at a temperature between and 60 C. the azide ofpantothenic acid with member of the class consisting offl-mercaptoethylamine and bis(,8-aminoethy1) disulfide.

8 3. Process for obtaining a compound of the formula,

which comprises reacting the ethyl ester of d-(+)-pantothenic acid withhydrazine to obtain the hydrazide of pantothenic acid, reacting saidhydrazide with nitrous acid to obtain the az'ide of pantothenic acid,and thereafter reacting said azide with B-mercaptoethylamine.

4. Process which comprises reacting at a temperature between 0 and C.the azide of pantothenic acid with a member of the class consisting offl-mercaptoethylamine and bis'(Baminoethy1)- disulfide.

5.'Process which comprises reacting at a temperature between 0 and 60 C.the azide of d-(+)-pantothenic acid with a member of the classconsisting of B-mercaptoethylamine and bis (p-aminoethyl) disulfide.

6. Process which comprises reacting at a temperature between 0 and 60 C.the azide of d-(+)-pantothenic acid with S-mercaptoethylamine.

7. Process which comprises reacting at a temperature between 0 and 60 C.the azide of d-(+) -pantothenic acid with bisqs-aminoethyl) disulfide;

ESMOND E. SNELL. EUGENE L. WITTLE. JAMES A. MOORE.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,340,757 Kaase et al. Feb. 1,194.4 2,485,116 Savard Oct. 18, 1949

1. PROCESS FOR OBTAINING A MEMBER OF THE CLASS CONSISTING OF A COMPOUNDOF THE FORMULA,